Method for cryopreserving blood vessels

ABSTRACT

A device for use in cryopreservation of blood vessels comprising a pair of stylets insertable into the ends of a dissected blood vessel segment. The stylets are mountable on a support track whereby the blood vessel can be distended and supported during cryopreservation procedures. Also disclosed is a freezing and thawing profile capable of maximizing endothelial cell survival. The use of chondroitin sulfate or similar compound is discussed as a novel cryoprotectant.

This is a division of application Ser. No. 088,092, filed Aug. 21, 1987.

TECHNICAL FIELD

The present invention relates to a device for stabilizing blood vesselsand more particularly to a preservation procedure for use duringfreezing blood vessels to ultra-cold temperatures whereby the bloodvessels can be preserved for extended periods of time. Also disclosed isa method utilizing the device for freezing and thawing of blood vessels.Cryopreserved blood vessels are useful for providing grafts to patientswho cannot provide their own blood vessel grafts or where fresh bloodvessels are unavailable.

BACKGROUND OF THE INVENTION

"Cryopreservation" is a technique for freezing and storing cellular andtissue matter such as blood vessels, which include veins and arteries,at extremely low temperatures while preserving the viability andfunction of the tissue. Each year, 360,000 small vessel coronary bypass"jumps" are performed in the U.S. alone. Another 100,000 peripheralvascular procedures, below the umbilicus, are also performed. Of thesmall vessel procedures, 15% are performed on patients who have alreadyhad a previous operation resulting in a lack of suitable availabletissue or on patients who are diabetic or have a disease which rendersthe tissue less than adequate. Clinically, the only alternative is touse less than optimal tissue or use artificial vessels which are proneto occlusion and thus are less than ideal. Because of the successesresulting from the cryopreservation of heart valve tissue (see copendingapplication Ser. No. 000,095 filed Jan. 2, 1987, which is incorporatedby reference in its entirety herein), and to date, more than 3,000cryopreserved valves and approximately 2,200 implants, it is theintention to expand this technology to vein and artery tissue as well.Thus, in the clinical setting, cryopreserved tissue would fill a needfor the aforementioned patents and would in addition lead to less traumafor the patient and reduce surgical time and expense.

Previous attempts at the use of allograft vessels have met with avariety of problems. The primary concern was inconsistency in the methodof harvest coupled with an inability to freeze and store the tissueproperly until its intended use. In addition, previous investigatorsfailed to perform the freezing process using state of the arttechniques, and consequently, the viability of the tissue was low andinconsistent and resulted in early loss of patency.

Although there have been a few published reports on the cryogenicpreservation of veins and arteries, there has been no publishedsystematic examination for the cryobiological variables involved in thepreservation procedure. Most investigators have simply infiltrated thevessel with dimethyl sulfoxide (DMSO) and rapidly frozen the tissue inliquid nitrogen. Several other investigators have used uncontrolled andunmeasured freezing rates. When dissected from the body, blood vesseltissue has a natural tendency to constrict. Investigations to date showthat under such conditions the endothelial lining of the vessel may bedenuded; therefore, if such a vessel is transplanted, it may be prone tothrombosis.

Preservation of the endothelial lining of these vessels is of particularimportance, because the internal endothelial lining of the blood vesselsactively inhibit thrombosis. Previous studies of saphenous veincryopreservation indicate that the major abnormality in the frozen andthawed tissue was destruction and loss of this tissue layer. A primarygoal of cryopreservation of the tissue is the prevention of ice crystalswhich damage or destroy cellular structure. Different freezing methodsare applicable to particular tissues; not all tissues are alike in theirability to withstand cryopreservation and thawing yet maintain effectiveviability. No investigator is known to have successfully applied thistechnology to the internal mammary artery or other arterial tissue.

SUMMARY OF THE INVENTION

The device of the present invention is a structure for supporting anddistending a blood vessel while permitting fluids to infiltrate thevessel to facilitate cryopreservation. More particularly disclosed is ablood vessel stent for use in cryopreserving blood vessels, the stentcomprising: first and second elongated stylets each having an endcapable of insertion within a portion of a blood vessel from a donor;means on said stylets operative to engage the interior of the bloodvessel and thereby facilitate fluid tight ligation of the blood vesselon the stylets; and support means receiving the stylets in selectivelyadjustable mutually confronting relation whereby the blood vessel isdistended between the stylets to prevent contraction of the bloodvessel, so that the stent supports the blood vessel through the stagesof procurement and cryopreservation. The method of the present inventionusing the device involves the technique for preparation of the vesselprior to stenting, removal, shipping to the processing laboratory,processing (including freezing), thawing and dilution. A particularemphasis is made for the preservation of the endothelium (inner lining)of the vessels in addition to keeping the basic integrity of the vesselwalls intact. This involves a "no touch" surgical technique coupled withvasodilation, the use of a stent, and the use of a unique freezingprofile that allows veins or arteries to be frozen down to thetemperature of liquid nitrogen, approximately -196° C., with minimaltissue damage due to ice crystal formation or osmotic shock. The presentinvention also includes a thawing schedule whereby the frozen tissue canbe rapidly thawed with minimal tissue damage. Vessels that arecryopreserved according to the present invention are alive when thawedand are ideally suited for replacing diseased or damaged vessels inpatients who for whatever reason do not have suitable vessels forcardiac or peripheral vascular reconstruction.

Accordingly, it is an object of the present invention to provide adevice and method for retrieval and handling of human or animal vein andartery tissue.

It is another object of the present invention to provide a device andmethod for preserving a living vessel for long periods of time.

It is yet another object of the present invention to provide a uniqueset of chemical constituents prior to the freezing process.

It is yet another method of the present invention to provide a devicefor supporting and distending a dissected blood vessel.

It is yet another object of the present invention to provide a uniquecooling schedule for freezing a vessel so that the vessel cells,including the endothelium, maintain high viability after it is thawed.

It is yet another object of the present invention to provide a methodfor cryopreserving a human vessel which allows rapid thawing of thevessel while maintaining maximum cell viability.

It is yet another object of the present invention to provide a livingvessel such that it is suitable for transplantation after long termstorage at super-cold temperatures.

It is yet another object of the present invention to provide a livingvessel with a specific and unique method for thawing.

These and other objects, features and advantages of the presentinvention will become apparent after review of the following detaileddescription of the disclosed embodiment and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a stent apparatus according to apreferred embodiment of the present invention.

FIG. 2 is an exploded isolated prespective of a stent apparatusaccording to a preferred embodiment of the present invention.

FIG. 3 is a schematic representation of a freezing profile for freezinga vein.

FIG. 4 is a representation of a thawing curve for a stented vein inapproximately 80-90 ml of solution according to a preferred embodimentof the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Referring now in more detail to the drawings, in which like numeralsindicate like parts throughout the several views, FIG. 1 shows a bloodvessel stent 10 which is composed of three main parts: a support track14, sliding proximal stopcock assembly 18, and fixed distal stopcockassembly 22. All three parts can be manufactured from any appropriatematerial which (a) is freezable to ultra low temperatures withoutadverse deforming or cracking, (b) maintains flexibility at ultra lowtemperatures, (c) is chemically inert and will not contaminate thevessel by leaching chemicals such as plasticizers into the vessel, and(d) can withstand and not react with dimethylsulfoxide ("DMSO"),ethylene or propylene glycol, glycerol or any other chemicals orsolvents used in the cryopreservation process. Track 14 is asubstantially straight elongated member used to mount stopcockassemblies 18 and 22. The track can be any convenient shape, but ispreferably a parallelepiped and has on one side a groove 26 running atleast part of its length for the purpose of stabilizing the stopcockassemblies 18 and 22 when mounted on track 14. On the surface at theproximal end of track 14 is located a ramp 30 with a flange having itslarger portion facing toward the distal end. Ramp 30 inhibits the mountfrom sliding off the end of track 14 once the mount is slid on thetrack.

Proximal slidable mount 34 is a cylindrical member having a recessedcylindrical opening axial at one end capable of matably receiving amounting pin. At the other end of the mount 34 is an opening radial tothe cylinder, capable of operatively receiving the track 14. Mount 34 isable to slide along track 14 so as to be adjustable for a given lengthof blood vessel segment.

Proximal stopcock assembly 18 includes a cylindrical mounting pin 38which removably mates coaxially within the axial hole of the proximalmount 34 and is preferably able to rotate about the cylindrical axis.Pin 38 of the proximal mount is orthogonally integrated with the body42, which is a hollow cylinder having a hole 46 extending through bothsides of the cylinder, positioned orthogonal to the pin. Along one endof body 42, positioned radially opposite each other are a pair ofprotrusions 50 which act as stops. Stopcock barrel 54, which fits withinthe cylindrical interior of the body 42, is a cylinder rotatablypositioned in a longitudinal orientation within body 42. A barrel hole58 is positioned perpendicular to the barrel 54 axis through the barreland selectively aligns with hole 46 in body 42 so as to allow apassageway through the stopcock. Lever 62 is a substantially rectangularmember perpendicularly integrated with stopcock barrel 54 at one end.When rotated so that the lever is parallel with track 14, barrel hole 58and body hole 46 are aligned to form a passageway through the stopcockwhile, when the barrel 54 is rotated clockwise to a positionperpendicular to track 14, the holes are not in alignment, therebyblocking the passage of fluid or air through the stopcock.

Stylet 66 is a hollow substantially straight elongated tube alignedparallel to the track 14, with a port 70 at the free end of the styletand with the other end of the stylet abutting the body 42. Stylet 66 isintegrated with body 42 and is orthogonal to the pin 38. Stylet 66 isaligned with and opposite hole 46. The hollow interior of the stylet 66communicates with the hole 46 in one side of the stopcock body 42. Onthe surface of stylet 66 is at least one and preferably two or moreforwardly tapering ramp surfaces 70 merging with stylet 66. Stylet 66 isdesigned to accommodate coupling with a vessel which may be press fitupon the stylet and can abut body 42. Stylet port 68 permits fluid orair to enter the blood vessel when the stylet 66 is coupled with theblood vessel. The stylet tube is sized so as to be essentially parallelwith the internal hollow bore of the tube. Ramp surface 70 terminates ina shoulder 74 which permits pressure-tight ligation as described morefully hereinbelow.

Fixed distal stopcock assembly 22 is essentially the same as slidingproximal stopcock assembly 18 except with the following notablemodifications. The bore of the axial mount hole is different than thatof the proximal mount hole and the diameter of the mounting pin 78 iscorrespondingly different from the diameter of pin 38 so that the twostopcock assemblies cannot be interchangeably mounted. This avoidsconfusion of which stopcock assembly is distal and which is proximal.Distal mount 82 is preferably permanently mounted on the distal end oftrack 14.

Distal body 86 also has integrated on its surface in alignment with thehole (as in hole 46) a Luer fitting 86 for detachably receiving asyringe. Alternatively, a hub 90 can be used without the Luer lockfitting for detectably receiving a fluid delivery device such as asyringe. Standard Luer fittings are commonly known to those skilled inthe art. In an alternative embodiment a hub 90 can be integrated in asimilar with the proximal stopcock assembly 18 to permit back flushingof the blood vessel before or after being mounted on track 14.

Distal lever 94 rotates in a manner similar but opposite to that ofproximal lever 62; i.e., when parallel to track 14 the distal lever 94is in the "open" position and when rotated perpendicular to the track isin the "closed" position. The stopcock levers are positioned so thatwhen both are parallel to track 14 they point in toward each other andwill not stretch the vessel during closure. In this way they do not addto the overall length of the structure, nor does the distal lever 94interfere with distal hub or Luer fitting 86 or 90.

The stent is used to support and distend a dissected vessel. Thedissection procedure is described in detail in the examples set forthhereinbelow. A specially designed perfusion solution containing asuitable vascular smooth muscle relaxant drug is applied along thelength of the vessel. After a period of time, usually about 10-15minutes, the vessel is carefully dissected using what is commonly termedas "no touch" technique, whereupon an appropriate incision is made onthe distal and proximal end of the vessel. If the donor's heart isbeating, the portion of the vessel which was transected will be tiedwith ligature. The removable stopcock assemblies 18 and 22 of stentapparatus 10 are placed one on each end of the vessel and a portion of aflushing/distention solution is perfused through the vessel. Thedissection can then be completed. The perfusion medium can be anyappropriate medium, preferably Delbecco's Minimal Essential Media("DMEM"). Other media include, but are not limited to, Medium 199, Eaglemedia, Hank's media, Delbecco's modified Eagle media, Iscoves modifiedDelbecco's media, Defined media A2, CMRL-1066, RPMI-1640 (also 1603,1630, or 1634), F10, F12, alpha media, or the like. To this media isadded serum such as but not limited to human serum, fetal calf serum("FCS"), serum substrate, or the like, and a vasodilator such as but notlimited to nitroprusside, dantrolene, nifedipine, verapamil,phentolamine, tolazoline, procardia, or the like. It is preferable touse papaverine having a concentration of from about 1×10E-4 to about30×10E-4M, more preferably about 3×10E-4M. This media solution also hascertain additives: bicarbonate, HEPES or similar buffer, glutamine,D-glucose and sodium pyruvate.

Before the vessel is completely removed, the stopcock ends are attachedto the support track 14 of the stent apparatus 10 in order that thevessel does not have the opportunity to contract. It is this naturalcontraction of the vessel that is deleterious to the inner endotheliallining of the vessel and for which the stent is designed to protect.Once the remaining portions of the vessel are dissected, the stent 10with the attached vessel is ready for transport in an outer container.

The stent 10 continues to provide support and protection for the vesselwhile progressing through other stages in the cryopreservation process.Upon arrival of the stented vessel for cryopreservation, the stent willprovide the support for continued flushing and inspection for tying ofcollateral vessels. During the freezing process the stent will be thesupport necessary to prevent the vessel side walls from collapsing andat the final phase during thawing and dilution the stent again keeps thevessel from collapse and facilitates the addition and removal ofcryoprotectant agents.

The present invention provides a method of freezing, storing and thawingendothelial lined tissue, such as a vein and artery. The tissue that isfrozen according to the present invention can be stored for long periodsof time at ultra cold temperatures with minimal loss of cell viability.The present invention includes a unique freezing profile that allows atissue such as vein and artery to be frozen down to the temperature ofliquid nitrogen, approximately -196° C., with minimal tissue damage dueto ice crystal formation or solution effects due to slow cooling. Thepresent invention also includes a thawing schedule whereby the frozentissue can be rapidly thawed with minimal tissue damage. Veins andarteries that are cryopreserved according to the present invention arebiologically viable when thawed and are ideally suited for replacingdiseased or mechanically damaged vessels.

The tissue to be preserved is only as good as that which is receivedinto the laboratory. Consideration must be given to donor age, healthand history of vascular disease. Another important consideration is thetime between death and the harvest of the vessels (warm ischemia) andthe time from the harvest of the vessels to laboratory processing (coldischemia). Attention must be paid to the method of handling the tissueduring procurement and the medium used to ship the tissue.

A donor that can be used as a source of human vessels which are frozenaccording to the present invention should be in the age range of up toabout 55 years of age and the donor should not have suffered fromsignificant atherosclerosis, diabetes, circulatory disorders, severehypertension, varicose veins, or communicable disease.

All procurement is to be performed under sterile conditions. Time delaybetween death and harvest will have a deliterious effect on theendothelial cell layer and therefore should be completed immediatelyafter expiration of the donor but in any case not longer than about 10hours post mortem. For example, the ideal length for coronary bypassprocedure would be to procure a vessel of approximately 17 cm with atleast a diameter of 4 mm. It is to be understood that other diametersand lengths are usable and are within the scope of this invention.

STERILIZATION

It was discovered that many antibiotics were extremely toxic to theendothelial layer of the vessels. This toxicity is the result of anumber of factors including time, temperature and mode of action. Inaddition to the antibiotics, the antimycotic (antifungal) agents may bedeleterious to the tissue endothelium. It is important to continuallytest new antibiotics and fungicides for cell toxicity and sterilizationefficacy, in order to improve cell viability and kill microbes resistantto previous agents.

A mixture of an antibiotic and an antimycotic were found to providesuitable sterilization results. A mixture of Imipenem and Ancoban werefound to be particularly suitable. Table 1 shows the effect ofantibiotic incubation on endothelial viability in vitro.

                  TABLE I                                                         ______________________________________                                        Effect of Antiobiotic Incubation on Endothelial Viability In Vitro            Antibiotic        Time    Significance                                        ______________________________________                                        APCVL              4 hrs.  NS*                                                Imipenem + gentamycin                                                                            4 hrs. NS                                                  APCVL             12 hrs. P < .05                                             PSA               12 hrs. NS                                                  Imipenem + gentamycin                                                                           12 hrs. P < .05                                             Imipenem-without  12 hrs. NS                                                  gentamycin                                                                    Imipenem + Ancoban                                                                              18 hrs. NS                                                  ______________________________________                                         *NS = Not Significant                                                         APCVL = Amphotericin B, 25 micrograms/ml, Polymixin B Sulfate, 100            micrograms/ml Cefoxitin, 240 micrograms/ml Vancomycin, 50 micrograms/ml       Lincomycin, 120 micrograms/ml                                                 PSA = Penicillin (501 U/ml) Streptomycin (50 mg/ml) Amphotericin B (10        mg/ml)                                                                   

FREEZING MEDIA

The medium in which the tissue is frozen is of great importance formaintaining a balanced cell environment. Time and temperature alsocontribute to whether a particular medium will be successful. Generally,a protein suspension, such as blood serum or artificial serum, must alsobe present for maximum cell viability.

A number of freezing media can be successfully used in practicing thepresent invention. Media, such as balanced tissue culture medium orsimple phosphate buffered saline, can be used for most tissue types. Forthis particular tissue type DMEM is the preferred medium with theassociated additive components discussed previously.

The freezing media is composed of the enriched DMEM plus FCS from about1% to 30%, more preferably 10% fetal calf serum; plus the range ofpapaverine discussed above, preferably about 0.012% papaverine; and,chondroitin sulphate having a concentration of from about 1% to 10%,preferably 2.5% to 5%, more preferably 2.5%.

Dimethylsulphoxide ("DMSO") is also added either in at least one step of1M or preferably in three steps of 0.25M, 0.5M and 1M titrations at 4°C. Concentrations of DMSO can range from about 0.5 to 3 molar. Theincrease in molarity of DMSO should preferably be gradual so as not totraumatize the blood vessel. DMSO can be added at higher temperaturesbut timing becomes far more critical and toxicity may result in sometissues.

An important innovation in endothelial protection used to further refineand preserve tissue integrity is to use chondroitin sulfate. Thisglycosaminoglycan (GAGS) is a major component of the extracellularmatrix. The molecular weight of chondroitin sulfate can vary from 5,000to 50,000 and it is a sulphated dissacharide consisting of repeat unitsof D-glucuronic acid and N-acetyl-D-galactosamine. Currently, thismaterial is an additive in K-sol, a solution used for the short term (4°C.) storage of corneas.

Examples of other suitable glycosaminoglycans include but are notlimited to hyaluronic acid, dermatan sulfate, heparin sulfate, heparin,and the like. Other cryoprotectants include but are not limited toglycerol, polyvinylpyrolidone, hydroxyethyl starch, and polyethyleneglycol, dimethylformamide, ethyl glycol, and the like.

Table II shows a series of experiments using this additive to thefreezing solution. Groups 1-6 used a protocol which differed from Groups7 and 8 in that a two hour 37° C. incubation was performed. The resultsindicate that the addition of chondroitin sulfate to the freeze mixturesignificantly improved endothelial viability.

These materials can be used in cryopreservation procedures with orwithout the stent apparatus 10. As a cryoprotectant chondroitin sulfateor its alternatives(GAGS) can be employed in procedures forcryoprotection of cells, tissues and organs in addition to bloodvessels.

                                      TABLE II                                    __________________________________________________________________________    Influence of Chondroitin Sulphate upon Endothelial Cell Viability                                       Cell Viability                                                                (Regression T-test                                                                      Treated/Control                           Chondroitin sulfate                                                                         (reps)                                                                            T   Signif                                                                            Control                                                                            Treated                                                                            Regression                                __________________________________________________________________________    (CS) addition (2.5 g %).sup.                                                  1)                                                                              Without vs with                                                                           6   3.309                                                                             **.sup.                                                                           (42) 51                                                                            (124) 174                                                                             2.95                                     chondroitin sulfate                                                           a) Saphenous veins only                                                                   3   3.426                                                                             *   (147) 95                                                                           (391) 429                                                                             2.66                                     b) Femoral veins only                                                                     3   4.091                                                                             *   (25) 29                                                                            (61) 71                                                                               2.44                                   2)                                                                              Without vs with CS                                                                        4    .457                                                                             ns  (217) 203                                                                          (187) 171                                                                          86                                          in mannitol during                                                            dilution (no CS during                                                        freeze)                                                                     3)                                                                              Without vs with CS                                                                        5    .992                                                                             ns  (109) 63                                                                           (60) 47                                                                            55                                          during 2 hr incubation                                                        (with CS during freeze                                                        and dilution)                                                                 a) Saphenous veins only                                                                   3    .606                                                                             ns  (94) 85                                                                            (89) 71                                                                            95                                          b) Femoral veins only                                                                     2    .603                                                                             ns  (83) 40                                                                            (30) 30                                                                               .36                                    4)                                                                              Unfrozen vs. frozen                                                                       4   12.171                                                                            **  (300) 327                                                                          (57) 56                                                                               .19                                      without CS, with CS                                                           during dilution and 2 hr                                                      incubation                                                                  5)                                                                              Without vs with CS                                                                        6   2.069                                                                             ns  69) 48                                                                             (32) 28                                                                            46                                          during all steps except                                                       no CS during freeze                                                         6)                                                                              Unfrozen vs with CS                                                                       2   5.752                                                                             **  (390) 457                                                                          (177) 163                                                                          45                                          during dilution, no CS                                                        during freeze and no                                                          incubation                                                                  Alternative CS Concentrations                                                 7)                                                                              Without vs with 10 g %                                                                    4   2.415                                                                             *   (118) 172                                                                          (319) 334                                                                             2.70                                     chondroitin sulfate (CS)                                                    8)                                                                              Without vs w/lg %                                                                         4   1.704                                                                             ns  (207) 131                                                                          (69) 82                                                                            33                                          chondroitin sulfate                                                         __________________________________________________________________________     .sup. ns = not significant; (P > .05);                                        * ≦P .05;                                                              ** ≦P .01                                                              .sup.  Groups 1-6 were performed using Protocol II. Protocol II differed      from Protocol I by the addition of postthaw 2 hr. 37° C. incubatio     in culture medium                                                        

FREEZE PROFILE

The freezing profile is of critical importance to successfulcryopreservation of a tissue. A multitude of variables exist to maximizetissue survival. For instance, the volume of fluid, the size of thetissue, geometry of the package and the combination of characteristicsincorporating cryoprotectant, tissue, and freezing media all contributeto an optimal freezing profile. It is to be understood that the priorart freezing profiles available for cell suspensions may not be suitablefor freezing blood vessels, and that prior art freezing profiles forheart valve tissue also may not be suitable. It has been determined thateach tissue has its own unique and optimal freezing profile. Thefreezing profile required to successfully cryopreserve one tissue may bedifferent from the freezing profile required to successfullycryopreserve another tissue.

A number of factors need to be considered when freezing a tissue. Amongthese factors are: the temperature around the equilibrium point,(generally +4° C., to the temperature at the freezing point); releaseand control of the exothermic heat given off at the freezing point;optimum cooling rate as which is determined by the permeability of thecell membrane to water; the surface to volume ratio of the cells; thetype and concentration of cryoprotective agents in the media;temperature and time of exposure to those agents, cooling rate removalof the cryopreserved tissue from the controlled rate freezer andimmersing the tissue into a liquid nitrogen refrigerator; and, warmingrate and the thickness of the tissue.

DETAIL OF FREEZING PROFILE

Thus, the method of harvesting veins from a donor, placing the vesselinto a medium with the proper tissue preserving characteristics fortransportation, and the use of proper cryopreservation agents prior tothe freezing of the vessel according to a freezing schedule is desirablefor proper cryopreservation. To accomplish this the chamber temperatureand cooling rate is controlled so as to produce the desired effect onthe sample. Since the blood vessel tissues cool at slightly differentrates, and phase changes occur at particular temperatures, carefulcontrol over the rates of freezing should be maintained.

The range of the freezing rate is also a function of the fluid volume inthe package containing the blood vessel as well as the geometry of thepackage. While the freezing profiles described herein are related to thevolume and geometry of the package, it is to be understood that thepresent invention encompasses those modifications of package designwhich result in a change in the volume and geometry, which in turn,result in a variance in the freezing rate. Freezing rates can vary to acertain amount at a given temperature. A suitable range is from about0.01°-100° C./min, about 0.1°-30° C./min, preferably about 0.3°-1°C./min and more preferably 0.5°-1° C./min. In a preferred embodiment ofthe present invention, the overall rate of freezing of the blood vesselis kept at approximately 0.5° C. per minute. A preferred freezingschedule used to cryopreserve the veins and arteries in the presentinvention is comprised of placing the packaged tissue, having a totalvolume of about 2.5 cm×25 cm, into a freezing apparatus. A typicalprofile for a specific fluid volume of 75-85 ml would have the initialtemperature of the chamber set to -10° C. The chamber is set to cool ata rate of 0.01° C./min. until the sample(s) reaches +4° C. At this pointthe tissue cools at a rate of 0.5°±0.2° C./min. until the sample reaches-2°±0.5° C., at which time a phase change is initiated. At this point,in order to prepare for the exothermic heat of fusion, the cooling rateis increased to -30° C. until the chamber reaches -70° C. Immediatelyafter the chamber reaches -70° C., the chamber is warmed at a rate of20° C./min. until the chamber reaches -60° C., whereupon thistemperature is held for a period of 17 minutes. During this time, theactual rate of vessel cooling is approximately -0.5°±0.2° C./min. At theend of this 17 minute period, the chamber is again warmed at a rate of10° C./min. until the chamber reaches a level of -30° C. The level of-30° C. is held for one minute and then the cooling of the chambercommences at a rate of 0.01° C./min. until the sample reaches -20° C.During this time, the actual rate of cooling of the sample isapproximately 0.5°±0.2° C./min. The final rate adjustment step is tocontinue cooling at 0.5°±0.2° C./min. until the sample reaches -65° C.or below. The result of this freezing profile is a rate of freezing fromthe start of the procedure until the end of about 0.5°±0.2° C./min. Thisrate of cooling has been optimized for vein tissue. The packagecontaining the vein is removed from the chamber and placed in thestorage liquid nitrogen refrigerator at -196° C. FIG. 3 illustrates atypical freeze profile of the present invention.

At such time that the vessel is requested by an implanting institution,the tissue will remain in the liquid nitrogen refrigerator.

SHIPPING

At the request of the implanting hospital, the tissue may be returned ina suitable insulated shipping container such as the container disclosedin U.S. Pat. No. 4,597,266 which is incorporated by reference in itsentirety herein, which includes a cardboard container with four inchesof foam insulation. A preferred embodiment is the use of dry ice whichhas been stored in liquid nitrogen whereupon liquid nitrogen infused dryice is placed around the package containing the vessel. The package isthen placed in the box. Appropriate protocols and other papers necessaryto document clinical implants are included in the shipment.

Upon arrival at the hospital, the vessel and its associated package areplaced into a liquid nitrogen freezer. The tissue cannot toleratestorage at temperatures above 130° C. since repeated cycling oftemperatures has a tendency to lessen the viability of the cells.Storage at temperatures equivalent to dry ice (-78.6° C.) is notconsidered sufficiently cold to prevent enzyme molecular degradation ofthe tissue and thus the storage time is significantly reduced.

THAWING

The thawing and diluting steps with an allograft must be clearlydefined, since ice crystal growth and osmotic shock can still harm thetissue. Venous blood vessels should be thawed by being placed in a warmwater bath. It has been determined that a thawing rate of 1°-1000°C./min., preferably, 10°-50° C./min. is appropriate for these vessels,depending upon the volume of the sample. Once thawed, the cryoprotectantof choice must be removed, usually in a step-wise fashion, to lessen theeffects of osmotic shock to the cells and thus allow for an orderlyequilibration of the cell with the surrounding medium. Time andtemperature are major considerations.

Immediately prior to the time that the vessel is to be used, it is to bethawed and the cryopreservation additives are to be removed using agradual dilution procedure to minimize osmotic damage. This thawing anddilution procedure is considered as important as the actual freezingprocedure since ice crystal formation can occur during this phase of theprocedure as well. In addition, inattention to proper temperature andtiming can and will either reduce the number of viable cells due to thetoxicity of the cryoprotectant ingredients and can on occasion actuallylead to the cracking of the vessels into unusable pieces.

The following specific examples will illustrate the method of thepresent invention as it applies to harvesting, freezing to ultra-coldtemperatures, and thawing of a blood vessel. It will be appreciated thatthe example will be apparent to those of ordinary skill in the art andthat the invention is not limited to this specific illustrative example.

EXAMPLE 1

The dissection is performed using sterile "no-touch" technique. Thevessel and adventitia are bathed in perfusion medium (DMEM, 10% fetalcalf serum and 0.12 mg of papaverine/ml) throughout the procedure. Thismedium also has as an additive: 25 mmol Hepes buffer, glutamine, 1000 mgD glucose/L and sodium pyruvate at pH 7.3±0.5(GIBCO Lab. Cat.#380-2320). Following removal of the adventitia, a caudal venotomy ismade and the stopcock assembly is inserted and ligated into place. Thevessel is gently perfused with the perfusion medium. The collateralvessels are identified and ligated approximately 1-2 mm from the mainvessel.

Before complete excision of the vessel, the support track 14 of thestent apparatus 10 is affixed to the distal and proximal stopcockassemblies 18 and 22. The dissection is completed, the proximal stopcockis closed and the vessel is infused with the perfusion medium up toapproximately 100 mmHg, whereupon the distal stopcock is closed in orderto keep the vessel distended. The vessel with the remaining perfusionsolution is placed into a shipping container such as a plastic tube anddouble sterile wrapped. The transportation box is commonly made ofstyrofoam and the tube containing the vessel is placed in water and iceat approximately 4° C. Hereafter, a courier service is commonly used tospeed the delivery to the laboratory, since the vessel, in order toremain living, should arrive within about 24 hours after the cessationof the donor heartbeat.

Upon arrival at the processing laboratory, the vessel is checked forproper packaging to verify that ice is still present. In a clean room, asterile field is established to inspect the vessel and complete theprocessing steps, the first of which is to inspect and trim extraneoustissue. After all collateral branches have been checked and there are noleaks, the stented vessel is ready to start antibiotic sterilization.

STERILIZATION In order to prophylactically sterilize the vessels, thefollowing critical procedure must be observed.

1. Imipenem (12 μg/ml) is placed into a solution of DMEM, which is atissue culture media;

2. Ancoban (antimycotic) (50 μg/ml) is also added to the solution;

3. the vessel is perfused and bathed in this solution is placed into a37° C. incubator for four hours.

Following the titration of the cryoprotectant, the vessels are packagedin pouches which are capable of withstanding the rigors of ultra coldcryopreservation. Normally, several successive layers of packaging areused in order to preserve sterility of the inner package containing thevessel. Finally, the vessel is ready for cryopreservation.

The freezing medium is composed of the enriched DMEM+10% fetal calfserum+0.12 mg/ml papaverine+2.5% chondroitin sulphate+IM DMSO or othersuitable protectant.

DETAIL OF FREEZING PROFILE

The freezing schedule used to cryopreserve the veins and arteries in thepresent invention is comprised of placing the packaged tissue with 75-85ml of fluid volume and cylindrical shape of 2.5 cm×25 cm into a suitablefreezing apparatus (such as Cryomed Model #1010 (990 C.)). Temperaturesprovided are approximate and a certain amount of latitude must beprovided to account for machine and instrument variance. The initialtemperature of the chamber is set to 10° C. The chamber is set to coolat a rate of 0.01° C./minute until the sample reaches +4° C. At thispoint the tissue cools at a rate of 0.3°±0.2° C./min. until the samplereaches -2° C., which initiates a phase change. At this point, in orderto prepare for the exothermic heat of fusion, the cooling rate isincreased to -30° C. until the chamber reaches -70° C. Immediately afterthe chamber reaches -70° C., the chamber is warmed at a rate of 20°C./min. until the chamber reaches -60° C., whereupon this temperature isheld for a period of 17 minutes. During this time, the actual rate ofvessel cooling is approximately 0.5°±0.2° C./min. At the end of this 17minute period, the chamber is again warmed at a rate of 10° C./min.until the chamber reaches a level of -30° C. The level of -30° C. isheld for one minute and then the cooling of the chamber commences at arate of 0.01° C./min. until the sample reaches -20° C. During this time,the actual rate of cooling of the sample is approximately 0.5°±0.2°C./min. The final rate adjustment step is to continue cooling at0.5°±0.2° C./min. until the sample reaches -65° C. or below. The resultof this freezing profile is a rate of freezing from the start of theprocedure until the end of 0.5°±0.2° C./min. This rate of cooling hasbeen optimized for vein tissue. The package containing the vein isremoved from the chamber and placed in the storage liquid nitrogenrefrigerator at -196° C.

Until such time that the vessel is requested by an implantinginstitution, the tissue will remain in the liquid nitrogen refrigerator.

SHIPPING

The frozen vessel is shipped in an appropriate container in the manneras described above.

THAWING

The thawing and dilution procedure can be performed as follows:

1. In a sterile field and with all sterile components, at least twoliters of sterile water warmed to about 37°-42° C. are placed into aninstrument tray or other vessel to accommodate the length of the stentedtissue.

2. The packaged vessel is removed from the protective cardboard box andplaced into the water bath. The package is to remain in this bath, whereit is manually or automatically agitated for approximately eightminutes, or until such time that gentle palpation of the package revealsthat no further ice crystals are present.

3. Once it has been determined that no ice crystals are present, thepackage is removed from the bath and the outer foil package is carefullywiped dry in the area between the two notches and with a pair ofscissors, the foil package is cut between the two notches. Sterileforceps are used to retrieve the inner clear pouch from the foil pouchand the process is repeated using sterile scissor to cut the innerpouch.

4. The stented vessel is carefully removed and placed into a cleansterile instrument tray or other suitable container whereupon the firstof the dilution steps takes place. A syringe is used to add to thevessel 50 cc of a solution ("Bottle A") containing:

0.5M mannitol;

10% fetal calf serum; and

DMEM.

The vessel is gently perfused with this solution and the washoutmaterial is allowed to rest in the tray whereupon the vessel is allowedto soak for five minutes. Instead of mannitol, any non-permeablebiocompatible sugar can be substituted, such as but not limited to,sucrose, sorbitol, trehalose, glucose or the like. The dilution of DMSOconcentration should be in decreasing steps of no more than 1/2 themolarity of the previous step. Thus, if the original DMSO concentrationis 1M, the first dilution step should be 1/2M sugar, followed by 1/4Msugar and finally zero molar sugar.

5. At the completion of step 4, the solution in the tray is discardedinto another suitable container and the following is added to the trayand mixed:

50 cc remaining in the bottle A from step 4; plus 50 cc from a solution("Bottle B") containing:

10% fetal calf serum; and

DMEM

The mixture is now 0.25M mannitol. A syringe is used to gently perfusethe vessel with this mixture, and it is bathed in this solution forapproximately 5 minutes.

6. At the completion of step 5, the contents of the tray are againdiscarded and the remainder of bottle B is added to the tray. The vesselis gently perfused and agitated in this solution for five minutes.

7. The vessel is now ready for transplant, but should not be removedfrom the stent until just prior to its intended use.

FIG. 4 illustrates a typical thawing curve based upon an 85 ml (1MDMSO+DMEM) sample containing a 20 cm. segment of vein being placed in 5liters of 42° C. water and package was agitated by hand twice per minutefor 15 seconds for each agitation until the vessel is thawed. The waterbath was allowed to cool as the thaw progressed. The thawing rate wasapproximately 25° C./min. A typical thawing time is about eight minutes.

TRANSPLANT

The vessels preserved by the method just described are intended for useas arterial substitutes for the coronary arteries or for peripheralvascular reconstruction. Accordingly, since the graft tissue isantigenic tissue several precautions and recommendations are suggested:

1. The donor/recipient blood groups should be compatible;

2. A postoperative course of antiplatelet therapy may include but not belimited to Dipyridamole or aspirin; and

3. Low dose short duration immunosuppression which may include but notbe limited to cyclosporin, prednisolone, and azathioprine to minimizethe possibility of graft rejection. By use of the above technique,platelet deposition is depressed, thus lessening the possibility forthrombus formation and eventual loss of patency. Other forms of vesselpretreatment to lessen immunologic effects such as incubation in animmunosuppressive agent could be considered.

STUDIES TO SUPPORT THE CLAIM THAT VESSEL ENDOTHELIAL CELLS ARE PRESERVED

An endothelial cell line was comprised of clonal bovine endothelium(BFA-1c). These cells were cryopreserved in situ while attached toplastic tissue culture substrate. Viability was determined by using acombination of acridine orange and propidium iodide, a dyeinclusion/exclusion assay. Dimethylsulfoxide was found to be moreeffective than other cryoprotective agents tested including glycerol,hydroxyethyl starch and polyvinylprolidone. It was determined that 1MDMSO was optimal when used in combination with slow cooling rates. SeeTable III.

                                      TABLE III                                   __________________________________________________________________________    Studies on Freezing Procedures for Veins                                      (using limiting dilution assay on endothelial cells)                                                   1Cell Viability                                                               (Regression) T-test                                                                     Treated/Control                            Freezing rates                                                                              reps                                                                              T  Signif.sup.1                                                                      Control                                                                            Treated                                                                            Regression                                 __________________________________________________________________________    1)                                                                              Unfrozen vs 0.5°/min,                                                              6   6.696                                                                            **  (258) 338                                                                          (153) 138                                                                          .59                                          3 step DMSO addition                                                        2)                                                                              Unfrozen vs 3°/min,                                                                3   3.554                                                                            *   (248) 228                                                                          (96) 56                                                                            .39                                          3 step DMSO addition                                                        3)                                                                              0.5° vs 3°/min,                                                             3   1.679                                                                            ns  (119) 95                                                                           (96) 56                                                                            .81                                          3 step DMSO addition                                                        4)                                                                              Without vs with DMSO                                                                      3   4.512                                                                            *   (20) 18                                                                            (109) 84                                                                           5.45                                         0.5°/min (3 step)                                                    __________________________________________________________________________     .sup.1 ns = not significant, p > 0.05                                         * = p ≦ .05                                                            ** = p ≦ .01                                                      

At 0.5° C./min., more than 70% of the endothelial cells survived, ascompared with a cooling rate of 10° C./min. or more where less than 20%of the cells survived. See Table IV.

                  TABLE IV                                                        ______________________________________                                        Influence of Cooling Rate Upon Survival of a                                  Bovine Endothelial Cell Line Cooling Rate °C./min                              Experiment #                                                                           1/2     3       10    30                                     ______________________________________                                        Viable Cells                                                                            1          68.8    58.7  17.5  15.3                                 (% Total) 2          78.9    63.1   3.1  16.1                                           3          76.7    56.4  17.5  10                                             4          80.3    28.4  24.5   5.3                                 Mean plus one         75.68   51.65                                                                               15.65                                                                               11.68                               standard             +/-     +     +/-   +/-                                  deviation             5.1     13.64                                                                               9.0   5.0                                 ______________________________________                                    

The previous experiment was repeated using actual saphenous vein vesselsand utilizing the protocol for preparation and titration of the DMSOdiscussed earlier. In essence, DMSO is added in three steps of tenminutes each at 4° C., such that the concentration increase from 1/4,1/2 to 1M. The DMSO is mixed with DMEM containing 25 mM HEPES buffer and10% fetal calf serum. The freezing rate was varied from either 0.5° or3° C./min. The experiments show that the freezing rate of 0.5° C.resulted in a higher percentage of endothelial integrity than the fasterrate of 3° C. See Table V.

                  TABLE V                                                         ______________________________________                                        Endothelial Integrity After Cryopreservation at 0.5° or 3°      C./min                                                                        Rate                  Endothelial Integrity (%)                               (°C./min)                                                                      Sample #  Vein #  Mean +/-se                                                                             % of Control                               ______________________________________                                        Control 114       38      86+/-1   --                                         3       10        4       62+/-7   72.1                                       0.5     20        7       70+/-5   81.4                                       ______________________________________                                    

We claim:
 1. A method of maintaining intact a layer of endothelial cellspresent in a blood vessel during cryopreservation comprising:i)contacting said blood vessel with an effective amount of acryoprotectant composition comprising a medium for freezing said bloodvessel, a cryoprotectively effective concentration of a cell penetratingcryoprotectant and a cryoprotectively effective concentration of aglycosaminoglycan; and ii) maintaining said blood vessel in contact withsaid composition at a temperature below -100° C.
 2. The method accordingto claim 1 wherein said composition further comprises an antibiotic. 3.The method according to claim 1 wherein the glycosaminoglycan ischondroitin sulfate.
 4. The method according to claim 1 wherein saidcell penetrating cryoprotectant is dimethylsulfoxide.
 5. The method ofclaim 1 wherein the freezing schedule comprises:(a) placing the bloodvessel into a freezing chamber; (b) cooling the sample at a suitablerate until the sample reaches about -2° C.; (c) cooling the sample aftersubstantial completion of phase change at a suitable rate; (d) removingthe blood vessel from the chamber; (e) placing the blood vessel in aliquid nitrogen refrigerator at a temperature of about -196° C.
 6. Themethod of claim 5 wherein the cooling rate is in a range of from about0.1°-100° C./min.
 7. The method of claim 6 wherein the cooling rate isin a range of from about 0.1°-30° C./min.
 8. The method of claim 7wherein the cooling rate is in a range of from about 0.3°-1° C./min. 9.The method of claim 8 wherein the cooling rate is in a range of fromabout 0.5°-1° C./min.
 10. The method of claim 9 wherein the cooling rateis in a range of from about 0.5° C./min.
 11. The method of claim 5wherein the freezing schedule comprises:(a) placing the blood vesselinto a freezing chamber, then (b) cooling the chamber from an initialtemperature of about -10° C. at a rate of about 0.01° C. per min. untilthe sample reaches about +4° C.; then (c) cooling the chamber at a rateof about 0.3° C. per min. until the sample reaches about -2° C.; then(d) cooling the chamber at a rate of about 30° C. per min. until thechamber reaches a temperature of about -70° C.; then (e) warming thechamber at a rate of about 20° C. per min. until the chamber reaches atemperature of about -60° C.; then (f) holding the chamber at atemperature of about -60° C. for 17 minutes; then (g) warming thechamber at a rate of about 10° C. per min. until the chamber reaches atemperature of about -30° C.; then (h) holding the chamber at atemperature of about -30° C. for one min.; then (i) cooling the chamberat a rate of about 0.01° C. per min. until the sample reaches atemperature of about -20° C.; then (j) cooling the chamber at a rate ofabout 0.5° C. per min. until the sample reaches a temperature of about-65° C.; then (k) removing the blood vessel from the chamber; and then(l) placing the blood vessel in a liquid nitrogen refrigerator at atemperature of about -196° C.